Intrigued by the essential role of mtDNA replication in selective inheritance, we carried out genetic screens for genes required for mtDNA replication. We reported a mitochondrial outer membrane MDI that recruits a translational stimulator, Larp to mitochondrial outer membrane. MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondria proteins including mtDNA replication factors. Lack of MDI abolishes mtDNA replication in the ovary and fails to limit the transmission of mt:coIT300I. This work not only illustrates a novel translational boost on the mitochondrial outer membrane essential for the massive mitochondrial biogenesis and mtDNA replication in the ovary, but also confirms that mtDNA replication mediates the selective inheritance. Our genetic screening also identified several mitochondrial RNA binding proteins including the mitochondrial RCC1 like protein encoded by CG3862 locus and a mitochondrial PPR repeats protein, both of which are essential for mtDNA transcription and replication. We are now trying to elucidate the molecular basis of how these protein influence mtDNA replication. mtDNA selection occurs at a late germarium stage where mtDNA replication preferentially occurs in healthy mitochondria. Interestingly, MDI protein is highly expressed at that stage, and acts together with Larp to activate mtDNA replication. We hypothesize that MDI-Larp complex might sense mitochondrial fitness, selectively enrich on healthy mitochondria and boost mtDNA replication. We found that Larp was recruited to mitochondria by MDI in control ovaries, however, it displayed a diffuse pattern after either FCCP treatment or in mt:CoIT300I ovaries at the restrictive temperature. On the other hand, the overexpression of Tom20-Larp that constitutively localizes on mitochondria surface regardless of mitochondrial fitness, abolished mtDNA selective inheritance in heteroplasmic flies. These observations strongly suggest that Larp is required for the selective proliferation of healthy mitochondria. We found that both MDI and Larp were hyper-phosphorylated on depolarized mitochondria. We are now testing whether phosphorylation on these proteins might destabilize the association between MDI and Larp, and impair mtDNA replication and selective inheritance. Our model predicts that the successful selection of individual organelle/genome demands effectively segregation of mtDNA into individual organelles, to minimize intra-organelle complementation. The lower the copy number of mtDNA per organelle, the more effective selection would be. It is now recognized that mtDNA is packed with various proteins into nucleoid structure that is essential for mtDNA transcription, replication and segregation. However, the structural organization and the molecular nature of mtDNA nucleoid remain largely unknown. We developed a genetic reporter, mitochondrially-targeted Dam methylase (MitoDam) that binds to mtDNA specifically. We are now using MitoDam to target APEX to mtDNA, will apply spatial labeling to comprehensively identify nucleoid proteins. This work would allow a systematically analysis of mtDNA organization, maintenance and segregation, and to test their role in mtDNA selective inheritance. mtDNA encodes essential subunits of the ETC complex. mtDNA mutations often disrupt the ETC complex and consequently render mitochondria less active. Hence, the functional expression of mtDNA might be a prerequisite for cell to recognize mtDNA mutations. We found that mitochondria in germline stem cells and proliferating cysts were not active, but became active in the 16-cell cyst prior to the initiation of selective mtDNA replication. It is therefore a conceivable hypothesis that mitochondrial activation (or mtDNA expression) might serve as a stress test, to distinguish healthy mitochondria carrying the wild type genome, from defective ones carrying mutated mtDNA. We have conducted an RNAi screen in the Drosophila ovary to identify cellular pathway required for mitochondrial activation. We found that knockdown of insulin signaling and dmyc in germ cells; and knockdown of dilp5 and wnt signaling in follicle cells impaired mitochondrial activation in germ cells. We are trying to order these signaling molecules into a functional cascade that leads to the mitochondrial activation, and testing whether these signaling pathways are indeed required for mtDNA selective inheritance. One factor hindering genetic analyses of mitochondrial DNA in metazoans is a lack of methods for mtDNA transformation. A successful procedure for mtDNA transformation requires three key steps: 1) introducing exogenous DNA into the mitochondria of living cells; 2) integrating exogenous DNA into the endogenous mtDNA; 3) selecting for recombined mtDNA. Mitochondrial-targeted restriction endonucleases (mtREs) have been used to target mtDNA containing the restriction site and selecting for mtDNA variants abolishing the cleavage sites, and to induce double strand breaks on mtDNA that promotes recombination. Therefore, the major technical hurdle toward successful mtDNA transformation is to effectively introduce DNA into mitochondria of living cells. We are now trying two approaches: a) bacterial conjugation mediated gene transfer, and b) lambda phage mediated DNA delivery to deliver engineer DNA into mitochondria and use mtREs to induce and select for recombinants.